Method of producing nucleosides by fermentation

ABSTRACT

All strains of Bacillus subtilis which produce inosine or guanosine by fermentation give higher yields of these nucleosides when the culture medium contains potassium, magnesium, or calcium ions in much higher concentrations than were commonly employed heretofore.

United States Patent lliroshi Shlbai;

Akira llama, both 01 Kanagawa-ken; Akio Yamanoi, Tokyo; Teruo Shiro, Kanagawaken; Kazumoto Kinoshita, Tokyo, all of [72] Inventors Japan [21] App]. No. 788,913

[22] Filed Jan. 3, 1969 [45] Patented Dec. 7, 1971 73] Assignee Ajinolnoto Co., Inc.

Tokyo, Japan [32] Priorities June 11, 1965 June 1 l, 1965, Japan, No. 40134333; June 27, 1966, Japan, No. 41/41685 Continuation of application Ser. No.

648,970, June 26, 1967, now abandoned Continuation-impart of application Ser. No. 554,322, June 1, 1966, now abandoned. This application Jan. 3, 1969, Ser. No. 788,913

[54] METHOD OF PRODUCING NUCLEOSIDES BY FERMENTATION 7 Claims, No Drawings [52] U.S. Cl 195/28,

195/ l 00 [51] v ClZd 13/06 [50] Field oiSearch 195/28 N [56] References Cited UNITED STATES PATENTS 3,111,459 11/1963 Motozakiet a1 195/28 N 3,222,257 l2/l965 Shiro et al 195/28 N Primary Examiner-Alvin E. Tanenholtz Auorney Kurt Kelman soning materials in large amounts. They may be produced by.

chemically or bichemically phosphorylating the corresponding nucleosides which are available by fermentation or by chemical synthesis.

Conventional nucleoside fermentation is carried outbyculturing a strain of bacteria capable of producing the nucleoside on a nutrient medium containing an assimilable carbon source, such as glucose; a nitrogen source, such as ammonium salts, nitrates or urea; one or more organic nutrients, such as amino acids, vitamins, adenine or a derivative thereof; and small amounts of inorganic nutrients, such as potassium dihydrogen phosphate, magnesium sulfate or ferrous sulfate, and by recovering the nucleoside produced and accumulated in the cultured broth.

An object of the present invention is to produce nucleosides in high yield by fermentation.

We have found that inosine and guanosine can be produced in very high yield when the nucleoside fermentation is carried out in a medium containing higher concentrations of potassium ions, magnesium ions or calcium ions than were used heretofore.

Potassium ion is necessary for the production of a nucleoside as well as essential for the growth of the micro-organism. When inosine fermentations were carried out in media containing varying amounts of potassium ions, the yields listed in the following tablel were obtained.

'KNO, was used.

Each 20 ml. batch of the medium employed contained Starch hydrolyzate (as glucose) 8 (g./dl.) (NI-1,) ,HPo, 0.08

M so, 711,0 0.04

FeSO; 71-1 0.001

MnSO, 4H O 0.001

Casein hydrolyzate 0.3

Ribonucleic acid 0.2

NH CL 1.5

KCL as listed in table 1 The several batches were placed in 500 ml. shaking flasks, sterilized at 1 C. for 10 minutes in an autoclave, and sterile CaCO was added to a concentration of 2.5 percent. The media were inoculated with Bacillus subu'lis M-582-R (ATCC No. 13953), cultured at C. for 70 hours, and the amount of inosine produced was determined.

' As is seen from the above table 1, the amount of inosine produced increases as the potassium ion increases to about 25 mg./dl., similar yields of inosine are obtained with 25 to 50 mg./dl. K A while conventional inosine fermentation medium contains about 10 to 20 mg./dl. of potassium ions. When about 500 to 2,500 mgJdl. of potassium ions are present, inosine is produced in a very high yield.

Analogous fermentations were performed with Bacillus subrills ATCC No. 19222 which is capable of producing guanosine, and the results listed in the following table 2 were obtained.

Table 2 Amount Concentration of KCI, of guanosine as K ions (mg.ldl.) produced (3.11.)

The medium contained, in g./dl.:

Starch hydrolyzate (calculated as glucose) 8 (NH,),HPO, 0.05

MgSO -7H O 0.04

FeSO -7H O 0.001

MnSO -4H O 0.001

Soybean protein hydrolyzate 4 Ribonucleic acid percent purity) KCl as indicated in table 2 Fourteen liter batches were placed in jar fermentors, sterilized at 115 C. for 15 minutes with steam, and inoculated with Bacillus subtllis ATCC 19222. The media were cultured at 34 C. for 68 hours with stirring and aerating. The pH of the media was maintained at 6.5 by introducing gaseous ammonia during the fermentation.

As can be seen from the above table 2, substantially similar guanosine yields were obtained in media containing 360 to 500 mg./dl. of potassium ions and in the medium containing the conventional 20 mg./dl. of potassium ions. However, when more than about 650 and up to 2,000 mg./dl. of potassium ions are present, guanosine is produced in a very high yield.

The potassium ions may be introduced into the medium by adding potassium hydroxide or in the form of potassium salts, such as potassium chloride, potassium nitrate or potassium phosphate. A relatively high concentration of phosphate ions in the medium results in a decrease in the nucleoside production, and potassium dihydrogen phosphate is preferably used in a concentration of not more than mg./dl. and supplemented by other potassium salts.

Magnesium ions are essential for the growth of the microoragnism, but only in a very'small amount. When 100 times the amount of magnesium required for growth is present in the culture medium, the inosine production may be markedly increased. As is shown in table 9 of example 3, a substantial growth of micro-organisms can be obtained with a magnesium ion concentration of 4 mg./dl. About 30 to 40 mg./dl. of magnesium ions are used in conventional inosine fennentation. We have found that inosine can be produced in a very high yield in a medium containing more than about 400 mg./dl. magnesium ions.

Similar results were obtained in guanosine fennentation, which was carried out in the same way as described above with reference to table 2, and are listed in the following table 3. Guanosine can be produced in very high yield in a medium containing magnesium ions in a concentration of more than about 230 and up to 800 mg./dl.

Table 3 Amount Concentration of MgCl as of guanosine Mg ions (mg.ldl.) produced (g/I.)

Magnesium ions may be added to the culture medium in the form of the magnesium salts, such as magnesium sulfate or magnesium chloride.

We have found that the nucleoside production can be greatly increased by elevated concentrations of calcium ions in a culture medium in which a strain of Bacillus subtilis capable of producing nucleoside is cultured on a carbon source, nitrogen source and other nutrients while the medium is being neutralized and the nitrogen source supplemented by gaseous ammonia or ammonia water during the fermentation.

The following table 4 lists the results of experiments on liter batches of a medium containing:

Starch hydrolyzate (as glucose) 8 (g./dl.)

KH PO 0.08

MgSO,-4l-l 0.001

adenine 0.035

soybean protein hydrolzate l NH CL as listed in table 4 CaCl as listed in table 4 Each batch was placed in a -liter fermentor, adjusted to pH 6.5 by gaseous ammonia introduced with air, inoculated with the inosine producing bacterium Bacillus sub- !ilis C-30-S (ATCC 13954), and cultured at 30 C. for 60 hours with stirring and aeration, while the pH of the medium was maintained at 6.5 by gaseous ammonia. The amount of inosine produced in each fermentor was determined and is listed in the following table 4.

NorE.IN: Amount of inosine produced. 7

Certain strains of Bacillus subtilis capable of producing inosine require chlorine ion for producing inosine, and accordingly ammonium chloride is used as a nitrogen source. Chlorine ions in the medium damage the apparatus. As is seen from table 4, the amount of chloride ion necessary for inosine production can be decreased by the presence of calcium ion in the medium.

ln order to obtain high yields of inosine, calcium ions should be present in a concentration of more than g./dl.

The following table 5 lists the results of experiments on media containing various amounts of calcium ions, and otherwise identical with those described with reference to table 2.

Table 5 Amount Concentration of CaCI, of guanosine as Ca ions (mgJdL) produced (g./|.)

As can be seen from the above table 5, in order to obtain a high yield of guanosine, calcium ions should be present in the medium in a concentration of more than about 370 mgJdl.

As can further be seen from the following table 6, the amount of chlorine ions necessary for satisfactory guanosine production can be reduced by the presence of calcium ions in the medium.

Micro-organisms which can be used in the present invention include Bacillus subtilis strains ATCC 19221, ATCC 19222, ATCC 13952, ATCC 13952 (M-582-R), ATCC 13954 (C-30- S), ATCC 13955 and ATCC 13956 (C-30). All members of the species Bacillus subtilis which produce insoine, guanosine or inosine and guanosine were found to give better inosine, guanosine or inosine and guanosine yields with higher concentrations of potassium, magnesium and/or calcium ions than with the amounts of these ions generally used heretofore.

The following examples further illustrate the invention.

EXAMPLE 1 Several batches of a culture medium containing 8 g./d1. of glucose, 0.08 g./dl. of diammonium hydrogen phosphate, 0.001 g./dl. of ferrous sulfate-heptahydrate, 0.001 g./dl. of manganese sulfate-tetrahydrate, 0.3 g./dl. of casein hydrolyzate, 0.03 g./d|. of adenine, 1.5 g./dl. of ammonium chloride and potassium chloride as listed below were adjusted to pH 7.2 with ammonia water, and each 20 ml. batch of the medium was poured onto a 500 ml. shaking flask. Each batch was sterilized at 1 10 C. for 10 minutes in an autoclave, and calcium carbonate which had previously been sterilized was added to a concentration of 2.5 g./dl. The batches were inoculated with Bacillus subtilis M-582-R (ATCC No. 13953), cultured at 30 C. for 72 or 94 hours, and inosine was produced as listed in table 7.

A stock culture and a seed culture were prepared as follows: Nutrient slants consisting of l g./d1. of peptone, 1 g./dl. of yeast extract, 0.3 g./dl. of sodium chloride and potassium chloride as listed in table 8 were prepared, one of the slants was inoculated with Bacillus subtilis C-30 (ATCC 13956) and cultured at 30 C. for 2) hours (stock culture). A fresh culture slant was inoculated from the stock culture, and cultured at 30 C. The culture obtained was inoculated on a fresh culture once more, and a seed culture was prepared.

Bacillus subtilis of the stock culture and the seed culture were inoculated on fermentation media having the same composition as in example 1 but diammonium hydrogen phosphate was replaced by 0.08 g./dl. of potassium Table 8 5 Culture slant K concentration in lnosine seeded Slant culture Fermentation produced (mg./dl.) medium (mg./dl.) (g./l.) I

Stock culture 0 0 7.]

Stock culture 2,100 0 7.3 Stock culture 4,200 0 8.9 Stock culture 0 1,570 8.9 Stock culture 2,100 L570 10.5 Stock culture 4,200 L570 10.5 Seed culture 0 0 6.9 Seed culture 2,100 0 6.7 Seed culture 4,200 0 9.3 Seed culture 0 L570 8.0 Seed culture 2,100 L570 8.5 Seed culture 4,200 1,570 10.4

EXAMPLE 3 I s s A culture medium cons1st1ng of: glucose 7 (g./dl.) N11,.c1. 1.5 KH PQ, 0.08 FeSO -4H O 0.001 141150 411 0 0.001 adenine 0.03 5 casein hydrolyzate 0.04 MgSO.,'71-1 O as listed in table 9 was prepared, the medium was adjusted to 7.4, and 20 ml.

batches of the medium were placed in 500 ml. shaking flasks. The media were sterilized at 110 C. for 10 minutes in an autoclave, and 2g./dl. of sterile CaCO was added to each medium. The media were inoculated with Bacillus subtilis C-30,

and cultured at 30 C. for 70 hours with shaking.

Table 9 Amount of MgSO added Amount of inosine EXAMPLE 4 Bacillus subtilis 030 (ATCC 13956) was inoculated on a medium as described with reference to table 4, but NH CL was replaced by KCl in the amounts listed in table 10, and the medium was cultured at 30 C. for hours with stirring and aerating. The results obtained are listed in table 10.

EXAMPLE 5 Culture media containing glucose 6 (g./dl.) (Ni-1., l-lPO 0.05 MgS0 -7H 0 0.04 FeSO -7H O 0.00l MnSO -4l-1 O 0.00l soybean protein hydrolyzate 1 adenine 0.035 NH,,CL 1 KC] as listed in the table 1 l were prepared, each medium was inoculated with Bacillus subn'lis No. 1346 (ATCC No. 13952), and cultured at 30 C. for 72 and 94 hours in the same procedure as in example 1. The amount of inosine produced is shown in the following table 1 1.

Table l 1 Amount of KCI Amount of inosine produced (g./l.) added as K ion 72 hour's 92 hours (mg./dl.) incubation incubation EXAMPLE 6 A culture medium containing 8 g./dl. of starch acid hydrolyzate (glucose equivalent), 0.05 g./dl. of KH PO 0.04 g./dl. of MgSO -7H O, 0.00l g./dl. of MnSO -4H O, 4 g./dl. of soybean protein hydrolyzate, 0.08 g./dl. of ribonucleic acid percent purity) and NH CL, KCI and MgCl as listed in table 12, was prepared, inoculated with Bacillus subtilis ATCC 19222, and cultured at 34 C. for 68 hours in a procedure similar to that described in example 1, whereby inosine and guanosine were produced in example 1, whereby inosine and guanosine were produced in the cultured broth as listed in IN: Amount olinosine produced .1.) GR: Amount of guanosine produced (g./l.)

EXAMPLE 7 Culture media each containing 8 g./dl. of glucose, 0.05 g./dl. of potassium dihydrogen phosphate, 0.04 g./dl. of mag- No'rE.-1N: Amount 01 inosine produced.

nesium sulfate heptahydrate, 0.001 g./dl. of ferrous sulfate heptahydrate, 0.001 g./dl. of manganese sulfate-tetrahydrate, 4 g./dl. of soybean protein hydrolyzate, 0.08 g./dl. of ribonucleic acid (80 percent purity) and 1.3 g./dl. of potassium chloride were prepared, the pH of the media was adjusted to 7.4, and each medium in an amount of 14 litres was poured into 22 litre jar fermentors. Each medium was sterilized at 1 15 C. for minutes, inoculated with Bacillus subtilis ATCC 19222, and cultured at 34 C. for 68 hours with stirring at 600 r.p.m. and with aeration at a rate of 2.6 I/minute. The pH of each medium was maintained at 6.5 throughout the fermentation by ammonia introduced with the air. Guanosine was produced and accumulated to a concentration of 4.6 g./l. in the cultured broth.

EXAMPLE 8 Guanosine was produced by fermentation as in example 7, but potassium chloride was replaced by 2 g./dl. of magnesium chloride hexahydrate, and guanosine was accumulated to a concentration of 5.0 g./l. in the broth during 66 hours of fermentation.

EXAMPLE 9 The potassium chloride was replaced in the procedure of example 7 by calcium chloride-dihydrate, the fermentation was performed otherwise in the same way as in example 7 for 66 hours, and 4.8 g./l. of guanosine was produced in the cultured broth.

EXAMPLE 10 The potassium chloride was replaced in the procedure of example 7 by 2.65 g./dl. of calcium chloride dihydrate, and Bacillus subtilis ATCC 19222 was replaced by Bacillus subtilis ATCC 19221, and the fermentation was performed for 70 guanosine, 6 g./l. of guanosine was produced.

What is claimed is:

1. In a process of fermenting an aqueous medium containing an assimilable carbon source, an assimilable nitrogen source, organic nutrients, and inorganic ions, which comprises inoculating said medium with a strain of Bacillus subtilis capable of producing at least one nucleoside of the group consisting of inosine and guanosine, and recovering said nucloeside, the improvement which consists of using an elevated concentration of potassium, magnesium, or calcium ions in said medium,

a. the elevated concentration of said potassium ions being between 500 and 2,500 mg./d1. if said nucleoside is inosine, and between 650 and 2,00 mg./d|. when said nucleoside is guanosine;

b. the elevated concentration of said magnesium ions being at least 400 mg./dl. when said nucleoside is inosine, and between 230 and 800 mg./dl. when said nucleoside is quanosine; and

c. the elevated concentration of said calcium ions being between l and 3 g./dl. when said nucleoside is inosine, and between 370 and 1,500 mg./dl. when said nucleoside is guanosine, said elevated concentration of calcium being maintained in said medium substantially from said inoculating to said recovering.

2. In a process as set forth in claim 1, said nucleoside being inosine.

3. In a process as set forth in claim 1, said nucleoside being guanosine.

4. In a process as set forth in claim 1, said strain being a member of the group of strains having ATCC numbers 13952, 13953,13954, 13955, 13956, 19221 and 19222.

5. In a process as set forth in claim 1, using said elevated concentration of said potassium.

6. In a process as set forth in claim 1, using said elevated concentration of said magnesium.

7. In a process as set orth 11'] claim 1, using said elevated 

2. In a process as set forth in claim 1, said nucleoside being inosine.
 3. In a process as set forth in claim 1, said nucleoside being guanosine.
 4. In a process as set forth in claim 1, said strain being a member of the group of strains having ATCC numbers 13952, 13953, 13954, 13955, 13956, 19221 and
 19222. 5. In a process as set forth in claim 1, using said elevated concentration of said potassium.
 6. In a process as set forth in claim 1, using said elevated concentration of said magnesium.
 7. In a process as set forth in claim 1, using said elevated concentration of said calcium, and maintaining said medium at a pH of not substantially less than 6.5 by addition of ammonia during said fermenting. 